This proposal aims to develop enhanced genetic analysis systems in the zygomycete fungus Mucor circinelloides. M. circinelloides is an opportunistic fungal pathogen that causes mucormycosis. The incidence of mucormycosis is increasing due to a rising number of immunocompromised individuals. M. circinelloides also serves as a model system to study light sensing and understand RNAi-mediated gene expression regulation. In addition, M. circinelloides is a candidate for biofuel production. However, the genetic analysis of this fungal system is limited due to 1) a limited number of markers for transformation; 2) inefficient transformation and low frequent homologous gene replacement; and 3) lack of ability to perform genetic segregation analysis. In the studies proposed within this document, we aim to enhance our ability for genetic analysis in this important fungal system by proposing three aims: 1) construction of a recyclable pyrG marker system; 2) enhancement of homologous recombination in gene disruptions; and 3) a pilot approach for mating and genetic analysis in M. circinelloides.